The present invention relates generally to enzymes for hydrolysis of sugars and particularly to an alkaline alpha-galactosidase which hydrolyzes a broad spectrum of galactosyl-saccharides such as melibiose, raffinose and stachyose and guar gum, at neutral to alkaline pH conditions.
The enzyme alpha-galactosidase (E.C. 3.2.1.22; alpha-D-galactoside galactohydrolase) catalyzes the hydrolysis of the terminal linked alpha-galactose moiety from galactose-containing oligosaccharides. These include, for example, the naturally occurring disaccharide melibiose (6-O-alpha-D-galactopyranosyl-D-glucose), the trisaccharide raffinose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside) and the tetrasaccharide stachyose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside). Alpha-galactosidases have potential use in various applications, and some examples are described by Margolles-Clark et al. (xe2x80x9cThree alpha-galactosidase genes of Trichoderma reesi cloned by expression in yeastxe2x80x9d, Eur. J. Biochemistry, 240:104-111, 1996). They may hydrolyze alpha-galactose residues from polymeric galactomannans, such as in guar gum; modification of guar gum galactomannan with alpha-galactosidase has been used to improve the gelling properties of the polysaccharide (Bulpin, P. V., et al., xe2x80x9cDevelopment of a biotechnological process for the modification of galactomannan polymers with plant alpha-galactosidasexe2x80x9d, Carbohydrate Polymers 12:155-168, 1990). Alpha-galactosidase can hydrolyze raffinose from beet sugar syrup, which can be used to facilitate the sugar crystallization from molasses, since the raffinose presents an obstacle to the normal crystallization of beet sugar (Suzuki et al., xe2x80x9cStudies on the decomposition of raffinose by alpha-galactosidase of moldxe2x80x9d Agr. Biol. Chem., 33:501-513, 1969). Additionally, alpha-galactosidase can be used to hydrolyze stachyose and raffinose in soybean milk, thereby reducing or eliminating the undesirable digestive side effects which are associated with soybean milk (Thananunkal et al., xe2x80x9cDegradation of raffinose and stachyose in soybean milk by alpha-galactosidase from Mortierella vinaceaxe2x80x9d Jour. Food Science, 41:173-175, 1976). The enzyme can also remove the terminal alpha-galactose residue from other glycans, such as the erythrocyte surface antigen conferring blood group B specificity. This has potential medical use in transfusion therapy by converting blood group type B to universal donor type O (Harpaz et al. xe2x80x9cStudies on B-anticenic sites of human erythrocytes by use of coffee bean alpha-galactosidasexe2x80x9d, Archives of Biochemistry and Biophysics, 170:676-683, 1975, and by Zhu et al. xe2x80x9cCharacterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytesxe2x80x9d, Archives of Biochemistry and Biophysics, 327:324-329, 1996).
Plant alpha-galactosidases from numerous sources have been studied and multiple forms of the enzyme have been described, such as in Keller F. and Pharr D. M., xe2x80x9cMetabolism of Carbohydrates in Sinks and Sources: Galactosyl-Sucrose Oligosaccharidesxe2x80x9d, In: Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 7, pp. 168-171, 1996, Marcel Dekker, Publ., N.Y. These can be classified into two broad groups, acid or alkaline, according to the pH at which they show optimal activity. Practically all studies of alpha-galactosidases have dealt with the acidic forms of the enzyme and these play important roles in seed development and germination. Alpha-galactosidases with optimal activity at alkaline pH are uncommon in eucaryotic organisms.
Alpha-galactosidases which show preferred activity to the disaccharide melibiose are often referred to as melibiases. These may have optimal activity at alkaline pH but are relatively specific to melibiose, with only little activity and low affinity to the trisaccharide raffinose. In addition, they characteristically function as a multimeric protein. For example, the bacterial alpha-galactosidase that has been described from Bacillus stearothermophilus (Talbot, G. and Sygusch, J., xe2x80x9cPurification and characterization of thermostable b-mannanase and alpha-galactosidase from Bacillus stearothermophilusxe2x80x9d, Applied and Environmental Microbiology, 56:3503-3510, 1990) has over a 15-fold higher activity with melibiose, as compared to raffinose and functions as a trimer. The alpha-galactosidase described from Escherichia coli K12 similarly has only about 4% of the activity with raffinose as compared to melibiose, with Km values of 60 mM and 3.2 mM, respectively, in addition to functioning as a tetrameric protein (Schmid and Schmitt, xe2x80x9cRaffinose metabolism in Escherichia coli K12: purification and properties of a new alpha-galactosidase specified by a transmissible plasmidxe2x80x9d, Eur. J. Biochemistry, 67:95-104, 1976). Similarly, the enzyme from Pseudomonas fluorescens H-601 (Hashimoto, H. et al., xe2x80x9cPurification and some properties of alpha-galactosidase from Pseudomonas fluorescens H-601xe2x80x9d, Agric. Biol. Chem., 55:2831-2838, 1991) has relative Km values for raffinose and melibiose of 17 and 3.2 mM, respectively, and functions as a tetramer.
There are obvious advantages to the use of a monomer protein with the desired enzyme activity, as compared to multimeric proteins. This has clearly been shown, for example, with the alpha-galactosidases from mung bean seeds (del Campillo, E., et al., xe2x80x9cMolecular properties of the enzymic phytohemagglutinin of mung beanxe2x80x9d, J. Biol. Chem. 256:7177-7180, 1981) in which the retrameric form of the enzyme disassociated into the monomeric form during storage, and this was accompanied by loss of activity.
The galactosyl-sucrose sugars, stachyose and raffinose, together with sucrose, are the primary translocated sugars in the phloem of cucurbits, which includes muskmelons, pumpkins and cucumber. The very low concentrations of raffinose and stachyose in fruit tissues of muskmelon suggest that galactosyl-sucrose unloaded from phloem is rapidly metabolized, with the initial hydrolysis by alpha-galactosidase, as described in xe2x80x9cCucurbitsxe2x80x9d, Schaffer, A. A., Madore, M. and Phan, D. M., In : Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 31, pp. 729-758, 1996, Marcel Decker Publ., N.Y.
P.-R. Gaudreault and J. A. Webb have described in several publications, (such as xe2x80x9cAlkaline alpha-galactosidase in leaves of Cucurbita pepoxe2x80x9d, Plant Sci. Lett. 24, 281-288, 1982, xe2x80x9cPartial purification and properties of an alkaline alpha-galactosidase from mature leaves of Cucurbita pepoxe2x80x9d, Plant Physiol., 71, 662-668, 1983, and xe2x80x9cAlkaline alpha-galactosidase activity and galactose metabolism in the family Cucurbitaceaexe2x80x9d, Plant Science, 45, 71-75, 1986), a novel alpha-galactosidase purified from young leaves of Cucurbita pepo, that has an optimal activity at alkaline conditions (pH 7.5). In addition to the alkaline alpha-galactosidase, they also reported three acid forms of the enzyme, and distinct substrate preferences were found for the acid and alkaline forms. Raffinose was found to be the preferred substrate of the acidic forms. The alkaline form had high affinity (Km=4.5 mM) and high activity (1.58 xcexcmol galactose formed per min per mg protein) only with stachyose. It had low affinity for (Km=36.4 mM) and low activity (0.14 xcexcmol galactose formed per min. per mg protein) toward the trisaccharide raffinose and hydrolyzed melibiose very slowly and therefore affinity and activity on that sugar was not calculated. Thus, this previously reported alkaline alpha-galactosidase can be described as having activity at alkaline pH but with only a narrow spectrum of substrates.
A further characteristic of the alkaline alpha-galactosidase from young leaves of Cucurbita pepo is that alpha-D-galactose, the product of the enzymatic reaction, is a strong inhibitor of the enzyme""s activity (Gaudreault and Webb, 1983), similar to many of the acid alpha-galactosidases. Geaudreault and Webb calculated that 6.4 mM galactose reduced the reaction velocity of alkaline alpha-galactosidase by 50%, in a reaction mixture containing 7.5 mM pNPG at pH 7.5. Such an inhibition by the product of the reaction (termed xe2x80x9cproduct inhibitionxe2x80x9d), generally has important physiological significance in metabolism.
Gaudreault and Webb (among others) have suggested that the alkaline alpha-galactosidase, as the initial enzyme in the metabolic pathway of stachyose and raffinose catabolism, was important in phloem unloading and catabolism of transported stachyose in the young cucurbit leaf tissue. It is likely that alpha-galactosidase similarly plays an important role in the carbohydrate partitioning in melon plants, and may have possible functions for phloem unloading in fruits of muskmelon. Recently, alpha-galactosidase activity at alkaline pH has been observed in other cucurbit tissue, such as cucumber fruit pedicels, young squash fruit and young melon fruit. Results obtained by Pharr and Hubbard (xe2x80x9cMelons: Biochemical and Physiological Control of Sugar Accumulation, In: Encyclopedia of Agricultural Science, vol. 3, pp. 25-37, Arntzen, C. J., et al., eds. Academic Press, N.Y., 1994) led them to suggest that stachyose degradation by alpha-galactosidase took place within pedicels of fruit of Cucumis sativus, especially in the regions where the pedicel joins the fruit. Recently, Irving et al. (xe2x80x9cChanges in carbohydrates and carbohydrate metabolizing enzymes during the development, maturation and ripening of buttercup squash, Cucurbita maxima D. Delicaxe2x80x9d, J. Amer. Soc. Hort. Sci., 122: 310-314, 1997) reported the developmental changes in alpha-galactosidase activities, measured at acid and alkaline pH, in buttercup squash (Cucurbita maxima) fruit. They found that at anthesis, alkaline activity was higher than activity at acid pH and that both activities declined during fruit development. Chrost and Schmitz (xe2x80x9cChanges in soluble sugar and activity of alpha-galactosidase and acid invertase during muskmelon (Cucumis melo L.) fruit developmentxe2x80x9d. J. of Plant Physiology, 151:41-50, 1977) reported approximately similar activities of alpha-galactosidase at acid and alkaline pH in Cucumis melo fruit at the anthesis stage.
However, all of these studies were carried out using the non-specific artificial substrate, p-nitrophenyl alpha-galactopyranoside (pNPG), which indicates alpha-galactosidase activity but does not reflect either the physiological role of the particular enzyme forms, or, more importantly, the substrate specificity of the particular enzyme. Thus, the prior art gives no reason to indicate that the above described alkaline alpha-galactosidase enzyme activity in the fruit pedicel or fruit tissue, which were assayed with pNPG, might in any way be novel.
Furthermore, it is well established that the artificial substrate pNPG often indicates a higher pH optimum for alpha-galactosidase activity than that which is observed with the natural substrates. For example, Courtois and Petek (xe2x80x9cAlpha-galactosidase from coffee beanxe2x80x9d, Methods in Enzymology, vol. 8:565-571, 1966) state that xe2x80x9cWith alpha-phenylgalactoside (pNPG) one observes a pH optimum at pH 3.6, and a second more pronounced peak at pH 6.1. Toward other substrates (melibiose, raffinose, planteose and stachyose) the pH curve is flatter, with a maximum between 3.6 and 4.0xe2x80x9d. Similar results were observed for the alpha-galactosidase of Vicia faba seeds (Dey. P. M. and Pridham, J. B., xe2x80x9cPurification and properties of alpha-galactosidase from Vicia faba seedsxe2x80x9d, Bioch. J., 113:49-54, 1969).
While it had been thought that alkaline alpha-galactosidase may be confined to the cucurbit family, which includes the above mentioned squash, cucumber and melon plants, it has recently been shown by Bachmann et al. (xe2x80x9cMetabolism of the raffinose family oligosaccharides in leaves of Ajuga reptens L.xe2x80x9d, Plant Physiology 105:1335-1345, 1994) that Ajuga reptens plants (common bugle), a stachyose translocator from the unrelated Lamiaceae family also contains an alkaline alpha-galactosidase. This enzyme was partially characterized and found to have high affinity to stachyose. Also, leaves of the Peperomia camptotricha L. plant, from the family Piperaceae, show alpha-galactosidase activity at alkaline pH, suggesting that they also contain an alkaline alpha-galactosidase enzyme (Madore, M., xe2x80x9cCatabolism of raffinose family oligosaccharides by vegetative sink tissuesxe2x80x9d, In: Carbon Partitioning and Source-Sink Interactions in Plants, Madore, M. and Lucas, W. J. (eds.) pp. 204-214, 1995, American Society of Plant Physiologists, Maryland). This indicates the possibility that alkaline alpha-galactosidases, including novel enzymes not previously described, may function in other plants that metabolize galactosyl-saccharides, in addition to the cucurbits.
The use of an acidic form of alpha-galactosidase in biotechnological and industrial applications presents problems. For example, the use of an acidic form of alpha-galactosidase to remove the galactose-containing oligosaccharides, which include raffinose and stachyose, from soybean milk presents a dilemma, as described by Thanaunkul et al., (xe2x80x9cDegradation of raffinose and stachyose in soybean milk by alpha-galactosidase from Mortierella vinaceaxe2x80x9dJour. Food Science, 41:173-175, 1976). The pH of soybean milk, which is 6.2-6.4, is well above the optimum pH range of the Mortariella vinacea enzyme, which is 4.0-4.5, as shown using the natural substrate melibiose. Lowering the pH of the soybean milk solution to conform to the acidic enzyme""s pH optimum caused the soybean proteins to precipitate and left a sour taste to the milk.
The use of alpha-galactosidase with an acidic pH optimum for the removal of raffinose from beet sugar faces a similar problem. In Suzuki et, 1969, (xe2x80x9cStudies on the decomposition of raffinose by alpha-galactosidase of moldxe2x80x9d Agr. Biol. Chem., 3-501-513, 1969) the pH of the beet molasses had to be lowered to 5.2 with sulfuric acid in order for the Mortariella vinacea enzyme to function.
Similarly, seroconversion of blood type B to blood type O would benefit from an alpha-galactosidase that is active at neutral to alkaline pH. since the described procedure (Goldstein et al., xe2x80x9cGroup B erythrocytes enzymatically converted to group O survive normally in A, B, and O individualsxe2x80x9d Science, 215:168-170, 1982) requires the transfer of centrifuged erythrocytes to an acidic buffer in order for the enzyme to function. Lowering the pH to the optimum for the coffee bean alpha-galactosidase caused the cells to be less stable and lysis to occur. Thus, the seroconversion is carried out at pH 5.6, xe2x80x9creflecting a compromise between red cell viability and optimal alpha-galactosidase activityxe2x80x9d, as reported in Zhu et al. (xe2x80x9cCharacterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytesxe2x80x9d, Archives of Biochemistry and Biophysics, 327:324-329,1996). Since the natural pH of blood is in the neutral to alkaline range (pH 7.3) alpha-galactosidase with activity in this pH range would have obvious advantages.
An additional limitation on the industrial utility of the currently available alpha-galactosidases is that their activity is frequently inhibited by the product of the reaction, galactose. As an example, the reported alkaline alpha-galactosidase from Cucurbita pepo leaves (Geaudreault, P. R. and Webb, J. A. xe2x80x9cPartial purification and properties of an alkaline alpha-galactosidase from mature leaves of Cucurbita pepoxe2x80x9d, Plant Physiol., 71, 662-668, 1983) is strongly inhibited by alpha-galactose and it was calculated that 6.4 mM galactose reduced the reaction velocity by 50%.
Thus, there is a well-established need for an alpha-galactosidase with high activity at neutral to alkaline pH and with activity towards a broad spectrum of natural galactose-containing saccharides, particularly, but not exclusively raffinose.
The present invention seeks to provide a novel alkaline alpha-galactosidase which hydrolyzes a broad spectrum of galactose containing compounds, including, but not limited to, melibiose, raffinose, stachyose and guar gum. A novel form of alpha-galactosidase (E.C. 3.2.1.22, alpha-D-galactoside galacrohydrolase) was isolated from young melon fruit mesocarp tissue, purified to homogeneity, as determined by SDS-PAGE gel electrophoresis, and characterized. The enzyme is characterized by optimal activity at neutral to alkaline pH (7-8), together with a broader substrate specificity, as compared to previously reported alkaline alpha-galactosidases. At minimum, the enzyme hydrolyzes stachyose, raffinose and melibiose and guar gum. By contrast, a previously described alkaline alpha-galactosidase, which is quite specific for the tetrasaccharide stachyose, shows low activity toward, and low affinity for, the trisaccharide raffinose and no detectable activity against the disaccharide melibiose. The novel alkaline alpha-galactosidase enzyme of the present invention was purified using techniques of differential protein precipitation, ion-exchange chromatography, gel electrophoresis under native and denaturing conditions. Its native molecular weight is estimated as 84 kDa and its denatured molecular weight is estimated as 79 kDa. It is not a glycoprotein, as determined by the absence of binding to the lectin Concanavalin A. It shows relatively low affinity to the inhibitor galactose (Ki=13 mM), together with relative insensitivity to the inhibitor. In particular, the enzyme has a high affinity for, and activity against the substrate raffinose.
These characteristics, particularly the neutral to alkaline activity optimum, together with the broad substrate specificity and most importantly the high affinity for raffinose, distinguish the enzyme from previously reported alpha-galactosidases. These very same characteristics, impart to this enzyme potential use in such diverse applications as the seroconversion of type B blood to type O blood, as well as a host of applications in the food products industry.
There is thus provided in accordance with a preferred embodiment of the present invention an enzyme isolated from an organism that metabolizes alpha-galactosyl containing saccharides, comprising an alpha-galactosidase (E.C. 3.2.1.22, alpha-D-galactoside galactohydrolase) with optimal activity in the neutral to alkaline pH range, and which hydrolyzes a variety of alpha-galactose containing saccharides, in particular raffinose. The enzyme is preferably a protein monomer and an exo-alpha-galactosidase.
In accordance with a preferred embodiment of the present invention the alkaline alpha-galactosidase is isolated from a plant that metabolizes alpha-galactosyl containing saccharides.
In accordance with a preferred embodiment of the present invention the alkaline alpha-galactosidase is derived from tissue of a member of the cucurbit family.
In accordance with a preferred embodiment of the present invention the alkaline alpha-galactosidase is derived from tissue of a melon plant.
Further in accordance with a preferred embodiment of the present invention the alkaline alpha-galactosidase is characterized by optimal activity in the range of pH 7-8.
Additionally in accordance with a preferred embodiment of the present invention the alkaline alpha-galactosidase is characterized by high affinity for the substrate raffinose and relatively low inhibition by galactose.
There is also provided in accordance with a preferred embodiment of the present invention a method for seroconversion of group B erythrocytes to group O erythrocytes, including providing an alkaline alpha-galactosidase which is hydrolytically active above about pH 7.0, and treating group B erythrocytes with the alkaline alpha-galactosidase so as to remove alpha-linked terminal galactose residues from the group B erythrocytes, thereby seroconverting the group B erythrocytes to group O erythrocytes.
There is also provided in accordance with a preferred embodiment of the present invention a method for reducing raffinose and stachyose levels in soybean milk.
There is also provided in accordance with a preferred embodiment of the present invention a method for reducing raffinose and stachyose levels in other plant products or tissues which contain these compounds.
There is also provided in accordance with a preferred embodiment of the present invention a method for modifying the rheological properties of galactose-containing gum products.
There is also provided in accordance with a preferred embodiment of the present invention a method for reducing raffinose levels in sugarbeet molasses thereby facilitating the crystallization of sucrose from said sugarbeet molasses.